We demonstrate, in the urethane-anesthetized rat, that within the medial prefrontal cortex (mPFC) there are clear subregional differences in the fast network oscillations associated with the slow oscillation Up-state. These differences, particularly between the dorsal and ventral subregions of the mPFC, may reflect the different functions and connectivity of these subregions. Cortical slow oscillations (0.1–1 Hz), which may play a role in memory consolidation, are a hallmark of non-rapid eye movement (NREM) sleep and also occur under anesthesia. During slow oscillations the neuronal network generates faster oscillations on the active Up-states and these nested oscillations are particularly prominent in the PFC. In rodents the medial prefrontal cortex (mPFC) consists of several subregions: anterior cingulate cortex (ACC), prelimbic (PrL), infralimbic (IL), and dorsal peduncular cortices (DP). Although each region has a distinct anatomy and function, it is not known whether slow or fast network oscillations differ between subregions in vivo. We have simultaneously recorded slow and fast network oscillations in all four subregions of the rodent mPFC under urethane anesthesia. Slow oscillations were synchronous between the mPFC subregions, and across the hemispheres, with no consistent amplitude difference between subregions. Delta (2–4 Hz) activity showed only small differences between subregions. However, oscillations in the spindle (6–15 Hz)-, beta (20–30 Hz), gamma (30–80 Hz)-, and high-gamma (80–150 Hz)-frequency bands were consistently larger in the dorsal regions (ACC and PrL) compared with ventral regions (IL and DP). In dorsal regions the peak power of spindle, beta, and gamma activity occurred early after onset of the Up-state. In the ventral regions, especially the DP, the oscillatory power in the spindle-, beta-, and gamma-frequency ranges peaked later in the Up-state. These results suggest variations in fast network oscillations within the mPFC that may reflect the different functions and connectivity of these subregions.NEW & NOTEWORTHY We demonstrate, in the urethane-anesthetized rat, that within the medial prefrontal cortex (mPFC) there are clear subregional differences in the fast network oscillations associated with the slow oscillation Up-state. These differences, particularly between the dorsal and ventral subregions of the mPFC, may reflect the different functions and connectivity of these subregions. These iAll procedures described below were independently reviewed in accordance with the UK Animals (Scientific Procedures) Act 1986 and the European Union Directive 2010/63/EU. Male Hooded Lister rats were supplied by Charles River Laboratories  and housed at Newcastle University’s animal facility in a temperature- and humidity-controlled environment consistent with the ARRIVE  guidelines. Rats were kept in an enriched environment (cage toys) under a 12-h light-dark cycle (lights on 7 AM-7 PM) with access to food and water ad libitum. Rats were housed in cages of two to four and were allowed a week of acclimatization before the experiment. Experiments were commenced ~2 h into the light (sleep) phase of the circadian cycle.Rats weighing 250–330 g were anesthetized with urethane . An initial dose of 1.5–1.9 g/kg was administered by intraperitoneal injection. Additional doses of 0.5 g/kg ip were given every half an hour until a deep anesthesia level  was achieved. through the dura, taking particular care not to damage the superior sagittal sinus that lies in the midline. The electrode was lowered in the z-plane using a one-axis oil-filled hydraulic micromanipulator .After a sufficient depth of anesthesia was achieved, the animal was fixed in a stereotaxic frame . A heating pad with feedback temperature control via a rectal probe  maintained the core temperature of the rat at 36.8°C. A pulse oximeter was attached to the animal’s hind paw and blood oxygen saturation was monitored . The animal breathed spontaneously, but to maintain an oxygen saturation of >90%, medical oxygen  was supplied through a tube mounted to the nose bar of the stereotaxic frame. A skin incision was made in the scalp and infused with lidocaine before the fibrous tissue and periosteum were retracted to expose bregma. A craniotomy was drilled above the mPFC of both hemispheres. Dual shank  16-channel silicon probes  were used for recording . Before insertion, the silicon probes were coated with the fluorescent dye DiI , dissolved in DMSO (1.5–2.5 mg/ml). The probes were implanted into the left and right mPFC Field potential recordings were made from two sites in each subregion of the mPFC: ACC, PrL, IL, and DP in both hemispheres. As each region contained two recording sites these are distinguished throughout as dorsal (d) or ventral (v) for each subregion. Post hoc histology confirmed that the shanks were equidistant from the midline so that the laminar position of the shank was in layer III of the mPFC in both hemispheres. Each of the 16 channels of extracellular signal passed through a unity-gain headstage  and was then amplified  and filtered (0.07–300 Hz for LFP) by a Plexon preamplifier. The LFP was digitized at 1,000 Hz and recorded on a PC (DELL Intel 4-core) running Plexon software (Sort Client).After the experiment the rat was killed by injection with Euthatal (200 mg/ml ip). The brain was removed from the skull and post fixed in 4% paraformaldehyde (PFA), 0.1 M phosphate-buffered solution (PBS) at 4°C for a minimum of 12 h before cryoprotection in 30% sucrose solution for a maximum of 24 h until it sank. Coronal sections (60–100 μm) were cut on a cooled vibratome  and collected in 0.1 M PBS. To visualize the cytoarchitecture, freely floating sections were then stained using either green fluorescent Nissl stain  or bisbenzimide H33258 (Sigma-Aldrich). After being stained, sections were mounted and coverslipped using Vectashield HardSet mounting medium .Photographs were taken using an Axio Imager Z2 microscope (Zeiss) and Axiovision 4.8 software (Zeiss). Tiled pictures were taken using the mosaic setting, with ×2.5 magnification. The rhodamine filter was used to reveal the DiI staining of the electrode tract. The fluorescein isothiocyanate (FITC) filter was used for the green fluorescent Nissl and the 4′,6-diamidine-2-phenylindol (DAPI) filter for the bisbenzimide stain. An overlay image was produced using Axiovison software.The raw LFP signal contained 50-Hz mains noise as well as higher frequency harmonics and was therefore notch filtered on all channels between 48 and 52, 98 and 102, and 148 and 152 Hz using a linear-phase finite impulse response filter (FIR) created using “fdatool.m” in combination with the MATLAB  “filtfilt.m” function. The filtering resulted in removal of a 4-Hz band from the gamma-frequency range of 30–80 Hz and a 6-Hz band from the high-gamma-frequency range of 80–150 Hz. In all cases this represented <10% of the frequency range contributing to those bands.All data analysis was performed offline using custom MATLAB (Mathworks) scripts. During slow oscillations, high-frequency oscillations occur transiently during the Up-state and are absent during the Down-state. Thus to investigate these nested oscillations in more detail it was first necessary to detect the UDS.t) was calculated using the Hilbert transform. The threshold to discriminate between the Up-state and Down-state was cos[ϕ(t) = 0]. To qualify as an Up-state the duration of the event had to exceed 300 ms and the average amplitude of the Up-state over all channels was required to be larger than 0.5 mV. To assess the oscillatory power with respect to the UDS, where the UDS cycles had different lengths, the instantaneous power was aligned to a “normalized” Down-state to Up-state cycle, as described below.UDS detection was performed using the phase of the slow oscillation, as described previously , except transition 1: Up-to-Down transition ; transition 2: Down-to-Up transition ; and transition 3: Up-to-Down transition . A phase vector was calculated that assigned −100 to the time point of transition 1, 0 to the time point of transition 2, and +100 to the time point of transition 3. The time points in between were filled with linearly spaced intermediate values, so that a phase vector was achieved with linear phase progression during the Down-state and linear phase progression during the Up-state. All UDS were then divided into 40 bins per state -, spindle (6–15 Hz)-, beta (20–30 Hz)-, gamma (30–80 Hz)-, and high-gamma (80–150 Hz)-frequency bands were calculated using a continuous wavelet transform on the LFP data B, using The amplitude of the slow oscillation was calculated by filtering the LFP (0.1-0.9 Hz) and aligning the trace to the normalized UDS cycle. The mean  of this UDS was calculated, and the slow oscillation amplitude was calculated as the peak-to-trough amplitude (maximum during Up state − minimum during Down state) of this average waveform.The cross correlation between two time series was calculated using MATLAB function “xcorr.m” and then normalized. The time lag between two signals was calculated as the time lag at the peak of the cross correlation. The cross power spectral density of the two signals was calculated using the MATLAB function “cpsd” with the following parameters: 30-s Hamming window, 50% overlap, and FFT length of 16,384. The phase angle was then calculated and plotted vs. frequency.All parameters were averaged from a continuous 120-s recording epoch within an animal yielding a mean value for each animal and each recording site. Statistical analysis was then performed on these mean values. In view of the small sample sizes normality could not be reliably assessed; therefore, nonparametric statistical methods were used. Group data are presented using box plots, in which the median is plotted as a line, the box constitutes the interquartile range (25–75 IQR), and the whiskers represented the most extreme values.2 with degrees of freedom (df). The Tukey’s multiple comparisons test was used for post hoc comparisons. The Wilcoxon signed rank test and Friedman’s test were performed in MATLAB (MathWorks). Data are reported as significantly different with P < 0.05, P < 0.01, and P < 0.001.A Wilcoxon signed rank test was used to compare two related samples with respect to one factor. Friedman’s test (one-way nonparametric ANOVA) was used to compare more than two related samples with respect to one factor and reported as χmethods). Post hoc histology confirmed that the shanks were equidistant from the midline so that the laminar position of the shank was in layer III of the mPFC in both hemispheres (methods), urethane evoked long-lasting (~1.5 - 2 h) periods of slow oscillations without spontaneous state changes.Bilateral field potential recordings were made from two sites in each subregion of the mPFC: ACC, PrL, IL, and DP in both hemispheres , and this was confirmed by cross correlation calculated between the slow oscillations in ACC and DP (Cii and Ciii), which showed no consistent dorsal-to-ventral time lag . Slow oscillations were also found to be highly synchronous across the two hemispheres (Dii and Diii) was not significantly different from zero . In addition, the cross spectrum phase analysis showed a phase difference close to zero between both the dorsal and ventral . The Up-state duration was significantly shorter than the Down-state duration (Aii and Aiii): median Up-state duration of 1.56 s  vs. a Down-state duration of 2.51 s .As the slow oscillations were synchronous across all mPFC regions data from just one channel in one region (ACC) was used to assess the frequency properties of the Up-Down states including, Up-Down cycle frequency, Up-state duration, and Down-state duration A. The frmethods) (2(7) = 6.25, Friedman’s test, P > 0.05, n = 8, 2(7) = 18.33, Friedman’s test, P < 0.05, n = 8, To examine whether the amplitude of the slow oscillation was consistent across mPFC regions we compared the amplitude recorded from two sites in each region B, measurmethods) C. The abA–C) present on the Up-state was compared between all subregions of the mPFC. For clarity, we predominantly describe data only from the left hemisphere recordings. However, the data for the right hemisphere recordings are shown in each figure for comparison, and their statistics are summarized in Next, we examined the fast oscillatory activity nested on the Up-state . Oscilla2(7) = 16.17, P < 0.05, Friedman’s test, n = 8], post hoc analysis did not reveal significant differences and the variation was smaller than for the other frequency bands. The latency to the peak delta power varied by mPFC subregion . Post hoc tests showed that latency to peak delta power was significantly shorter in ACC than DP but only in the left hemisphere .The power of the oscillations in the delta range did not show any consistent subregional pattern . AlthougA and B and see 2(7) = 37.62, P < 0.001, Friedman’s test, n = 8]. Post hoc tests showed that spindle power in the left dACC, vACC and dPrL was significantly greater (P < 0.05). This profile of dorsal-to-ventral area power changes occurred in all animals, and both hemispheres.The power of the oscillations in the spindle-frequency (6–15 Hz) range varied as a function of mPFC subregion with the highest power oscillations in ACC and PrL ( greater Bi than iA and C). In contrast, in DP the latency to peak spindle power was more variable, and spindle-frequency oscillations occurred predominantly in the latter half of the Up-state (A and C). Statistical analysis for the left hemisphere (2 (7) = 33.34, P < 0.001, Friedman’s test, n = 8]. Post hoc tests showed that latency to peak spindle power in ACC differed from DP and the latencies in the dPrL differed from the latencies observed in vDP . Similar dorsal-to-ventral differences were seen in the right hemisphere  = 48.96, P < 0.001, Friedman’s test, n = 8]. Post hoc tests showed beta-frequency power in the ACC and dPrL was significantly higher than in vIL and dDP. The beta power in the ACC was also significantly higher than in the dIL (P < 0.05 Tukey test). A similar profile was seen in the right hemisphere . Post hoc tests showed that the latency to peak power was significantly shorter in ACC than DP (Tukey test P < 0.05), with the same profile seen in the right hemisphere  = 52.88, P < 0.001, Friedman’s test, n = 8]. Post hoc analysis of the left hemisphere recording revealed that ACC differed from vIL and DP, dACC also differed from dIL, and dPrL differed from the dDP . A similar pattern of dorsal-to-ventral differences was also seen in the right hemisphere  = 32.42, P < 0.01, Friedman’s test, n = 8]. Post hoc tests showed that the latency to peak power was significantly longer in the vIL, dDP, and vDP than in the vACC  with, again, a similar pattern observed in the right hemisphere  = 47.25, P < 0.01, Friedman’s test). Post hoc tests showed that ACC and dPrL differed from vIL and DP and vPrL differed from dDP . Overall, similar dorsal vs. ventral area power differences were seen in the right hemisphere -frequency activity is known to depend on the activity of fast-spiking interneuron populations . The greThe different, but consistent, latencies for peak spindle, beta, and gamma power we have observed in the dorsal mPFC regions suggest that different neurons may be involved in generating the different types of activity. Our data also demonstrated that, in the dorsal mPFC regions, peak beta power occurred during a narrower part of the Up-state cycle compared with the gamma activity and at a shorter latency than the gamma-frequency peak power. Beta- and gamma-frequency activity may depend on different interneuron populations, that give rise to inhibitory synaptic potentials, with different decay time constants . The difUrethane is widely used to study sleep-related activity as several features of natural sleep, including spontaneous alternations in brain state ,b, and sWe demonstrate that the dynamics of Up-state associated fast oscillations show differences between dorsal and ventral mPFC. The higher power for all frequency bands in dorsal regions may be due to functional interactions between mPFC subregions in vivo through which dorsal regions may suppress the generation of fast oscillations in more ventral regions. Certainly there is evidence for strong interregional interactions in vitro , but furAbnormalities in sleep-related oscillations have been linked to a wide range of neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease  as well S. Gretenkord was supported by a Wellcome Trust Ph.D. Studentship 092994/2/10/2.No conflicts of interest, financial or otherwise, are declared by the author(s).S.G. performed experiments; S.G. analyzed data; S.G. prepared figures; S.G., A.R., M.A.W., S.E.G., and F.E.L. edited and revised manuscript; S.G., A.R., M.A.W., S.E.G., and F.E.L. approved final version of manuscript; A.R., M.A.W., S.E.G., S.G., and F.E.L. interpreted results of experiments; S.G. and F.E.L. drafted manuscript.


The effect of mTOR-Is on the overall incidence of tumors irrespective of their origin is not entirely clear. Furthermore, conflicting data have been shown on mortality under mTOR-Is.The current literature was searched for prospective randomized controlled renal transplantation trials. There were 1415 trials screened of which 13 could be included (pts. = 5924). A minimum follow-up of 24 months was mandatory for inclusion. Incidence of malignancies and patient survival was assessed in meta-analyses.The average follow-up of all trials was 40.6 months. Malignancy was significantly reduced under mTOR-Is compared to CNIs . This effect remained stable when combined with CNIs . When NMSCs were excluded the risk for malignancy remained significantly reduced under mTOR-I therapy (mono and combi) . Graft survival was minimally decreased under mTOR-Is . This effect was abrogated when mTOR-Is were combined with CNIs . Patient survival was not different .Posttransplant patients have a lower incidence of malignancy when treated with an mTOR-I no matter if it is used in combination with CNIs or not. This beneficial effect remains significant even when NMSCs are excluded. With currently used mTOR-I-based regimen patient and graft survival is not different compared to CNI therapies. The number of transplants and the alive transplant population is growing. In the US, an increase of 9.2% was encountered for annually performed transplants from 2010 to 2015 (OPTN data). This has implications for the medical system. According to an USRDS based analysis around 15.6% of renal transplant recipients are expected to die within the first three years after renal transplantation, of these 76.3% with a functioning graft representing 46.8% of all graft losses [Altogether, the incidence of post-transplant malignancies is increased 2- to 4-fold compared to the general population and tumors often show a more aggressive phenotype under immunosuppression –5. It isHence, big hopes were raised when anti-tumor activity could be attributed to mTOR-Inhibitors . Since tMirroring the complexity of this field the findings with respect to tumor incidence or recurrence have been inconsistent. Treatment of some tumor entities with mTOR-Is, i.e. Kaposi sarcoma, has been shown highly efficacious . In patiIn the meantime worrisome data have been published indicating a negative mTOR-I effect on patient survival –23.Altogether it remains unclear to this date if mTOR-Is should be used to reduce the overall incidence of cancer after solid organ transplantation.http://www.ncbi.nlm.nih.gov), ScienceDirect (http://www.sciencedirect.com) and the Cochrane Central Register of Controlled Trials  up to April 2017 using the following terms: mTOR-inhibitor OR sirolimus OR everolimus AND transplant AND malignancy or cancer. The search was performed by two reviewers .Full reports of controlled prospective trials were searched via PubMed  and ITT-analysis. These trials were required to have had at least two treatment arms, one with an mTOR-I based immunosuppression either with or without a CNI and one arm containing an mTOR-I free CNI-based immunosuppression. The mTOR-I had to be introduced within 3 months after the transplantation as this approach is commonly used in randomized trials. This selection criterion ensured a long follow-up under mTOR-Is with only minimal CNI exposure. The retrieved trials were screened for information on posttransplant malignancies, graft and patient survival. When several publications showed the same cohort of patients, the information was summarized. Trials were only included when a minimum follow-up of 24 months existed.Screening and inclusion of the articles was performed by two reviewers .2 statistic [metafor package in the statistical software package R (version 2.14.2). P values below 0.05 were considered significant and all confidence limits were on the 95% level.To summarize the available evidence, we calculated relative risks (RRs) for the incidence of posttransplant malignancies, graft and patient survival under CNI- and mTOR-I-based immunosuppression. If no malignancy was observed in a study arm 0.5 cases were added to both study arms to facilitate the calculation of the RR. If the incidence in both study arms was zero the incidence was set to 1% to receive a RR of 1. Publication bias was assessed by plotting study results against precision of the study (funnel plots) and the according regression tests . Betweentatistic . Accounttatistic . StandarThe following data were extracted from eligible articles by two reviewers : induction therapy, number of patients per treatment arm, mTOR-I dose, start of mTOR-I treatment post transplantation, graft and patient survival, trough levels, follow-up period, description, type and incidence of events of posttransplant malignancy, and statistical analysis of the posttransplant malignancy under mTOR-Is, mTOR-Is in combination with CNIs and CNIs.Methodological quality was assessed by three reviewers  using the Jadad score and Intention to treat (ITT) analysis , 28. TheThe literature search produced 1415 studies, of which 13 met the inclusion criteria. Thus, a total number of n = 5924 patients were included . The aveOnly longterm analyses on tumor incidence with and without NMSCs were performed. Furthermore, patient survival and graft survival censored for death were analyzed. The shortest follow-up duration was 24 months.Eleven of the 13 RCTs were considered to be of good methodological quality according to the Cochrane Collaboration’s tool  Figs and2 = 0.0%, Q-Test for heterogeneity: p = 0.69).Treatment with an mTOR-I  showed a significantly reduced risk for posttransplant malignancies compared to CNI treatment  . The reg2 = 0.0%, Q-Test for heterogeneity: p = 0.62).The analysis excluding NMSCs (n = 6) also revealed a reduced relative risk for the tumor incidence under mTOR-I treatment  . There w2 = 47.21%, Q-Test for heterogeneity: p = 0.12).RCTs with a combination therapy  showed a significantly reduced risk for the tumor incidence in comparison to CNI therapy  . There w2 = 0.00%, Q-Test for heterogeneity: p = 0.53).After exclusion of NMSCs (n = 2) there was also a significant difference  . There w2 = 0.0%, Q-Test for heterogeneity: p = 0.42).Taken together all studies on longterm tumor incidence , the risk of posttransplant malignancy was significantly reduced under mTOR-I treatment  . Heterog2 = 13.44%, Q-Test for heterogeneity: p = 0.54).When NMSCs were excluded 8 RCTs could still be included in the statistical analysis. Here, the relative risk was also significantly reduced under mTOR-Is  . There wThere were 7 RCTs included in this analysis. SIR was the mTOR-I used in 6 RCTs and EVRL in 1 RCT.2 = 18.22%, Q-Test for heterogeneity: p = 0.17).The ensuing analysis implied a minimal negative effect for the mTOR-I therapy even though a statistical significance was closely missed .There were four RCTs  which presented longterm graft survival data censored for patients’ death. The meta-analysis revealed an estimated combined RR of 0.99 .Taken together all studies with an mTOR-I-treatment arm (monotherapy or in combination with a CNI) compared to a CNI-based treatment , the overall graft survival was statistically superior under CNIs  . SimilarThere were 8 RCTs included in this analysis, which compared an mTOR-I- with a CNI-based treatment. Here, the mTOR-I was given without a CNI. SIR was the mTOR-I used in 7 and EVRL in 1 RCTs.2 = 0.00%, Q-Test for heterogeneity: p = 0.83).The mTOR-I showed a combined estimated RR of 1.00  compared with the CNI treatment . The reg2 = 0.00%, Q-Test for heterogeneity: p = 0.98).Five RCTs  presented longterm patient survival data in this setting. The meta-analysis revealed an estimated combined RR of 1.00 .When all studies with an mTOR-I-treatment arm either in monotherapy or in combination with a CNI were taken together and compared to a CNI-based treatment , the overall patient survival showed a RR of 1.00  were analyzed together.This was expected and in part shown by others before , 34 sincThus the more intriguing part was the next set of analyses for which NMSCs were excluded. For this, six studies could be included comparing mTOR-I without CNI vs. CNI. Another two trials with a comparison mTOR-I + CNI vs. CNI could be added.Taken all these analyses together the RR for malignancy w/o NSMCs was substantially and significantly reduced .Over the past decade a series of meta-analyses and large population-based trials tried to elucidate the mTOR-I anti-tumor effect and outline a certain pattern for cancer incidences after solid organ transplantation –9, 40.One of the first, Kauffman et al. published registry data from the OPTN/UNOS on this topic in 2005 . At thatIn the first meta-analysis on mTOR-I vs. CNIs, no significant difference with respect to tumor incidence could be reported . LimitinMore recently, a meta-analysis combined with registry-data showed a 51% reduction of NMSCs under a SRL-based compared to a CNI-based immunosuppression for renal transplant recipients while failing to show a benefit with respect to other tumors . Here, iSimilar data were reported by another meta-analysis on patients after renal transplantation receiving SRL . Here, NContrary to the above mentioned papers we tried to be more stringent about the study compilation including only trials with currently used mTOR-I regimen and comparable concentrations.A Collaborative Transplant Study (CTS)- analysis on n = 78146 first deceased-donor kidney transplant recipients with a mean follow-up of 4.2 yrs provided more detailed information on NMSCs . SimilarThe problems inherent to registry data are well known. And they may even be aggravated for informations which are not the primary focus of the registry. This was recently confirmed by a comparison of the SRTR with 15 linked cancer registries. Even though US transplant centers are required to report cancers in transplant recipients to the transplant network, this analysis revealed an extraordinary lack of accuracy and completeness for the SRTR . The autFor which tumor entities, other than NMSCs mTOR-Is have a beneficial effect we cannot tell by this analysis. Furthermore, we cannot exclude the possibility that mTOR-Is are permissive for some tumor entities while inhibiting others as has been postulated for prostate and renal cancers .de novo or an early “monotherapy” with an mTOR-I results in a higher percentage of BPARs and a high number of therapy dropouts [Our analyses found a statistically significant benefit for CNIs vs. mTOR-Is w/o CNIs. The RR was 0.99 and thus a clinical relevance questionable. For the combination of mTOR-Is and CNIs no such effect was seen. It has been repetitively shown that a dropouts –47. Thesdropouts –50 as isde novo (76%) and often with extraordinary high loading and maintenance doses. Importantly, mortality under “low dose” SRL, as is preferably used nowadays in transplantation was not increased.Another important aspect of our analysis is that there is no difference in patient survival (mTOR-I vs. CNI therapy). This is contrary to some reports of the recent years which indicated a potential threat for patients under mTOR-Is –23, 51. First, we did not have patient level information from the included RCTs. Second, malignancy and survival were the primary endpoint in some but not all of the RCTs. However, survival as the most important outcome variable has certainly been documented accurately. This most likely holds true also for malignancy events. Post-transplant malignancy and the as of yet unclear significance of an mTOR-I therapy is a highly relevant aspect. Third, the trials included patients with varying risk of malignancy: some excluded those with any cancer, while others excluded only those with a history of non-skin cancer.Early initiation or conversion to mTORI-I within 3 months of kidney transplantation may reduce the future risk of cancer, when compared with patients remaining on CNI-based regimens. The primary effect is against NMSCs but there also exists a significant effect against other tumors. The predominant part of the anti-tumor effect remains present even when administered in combination with a CNI. There is no increased mortality nor graft loss under currently used mTOR-I based regimen.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


P = 0.025) while no difference in the avidity of free anti-TF IgG was established. A negative correlation between the avidity of anti-TF IgG in tIgG and SNA binding in both groups was observed (P < 0.0001). The HAbs level negatively correlated with the anti-TF IgG avidity in tIgG only in donors (P = 0.003). Changes in the level of HAbs and Abs avidity showed a rather good stage- and gender-dependent diagnostic accuracy. Cancer patients with a lower anti-TF IgG avidity in tIgG showed a benefit in survival. Thus the TF-specific HAbs represent a particular subset of anti-TF IgG that differ from free serum anti-TF IgG in SNA reactivity, avidity, diagnostic potential, and relation to survival. Natural antibodies to the tumor-associated Thomsen-Friedenreich antigen (TF) are related to tumor immunosurveillance and cancer patients' survival. Hidden IgG antibodies (HAbs) to TF, their lectin reactivity, avidity, and clinical relevance were studied. HAbs were present in cancer patients and controls. A decreased level of IgG HAbs was detected in cancer. The HAbs level positively correlated with the sialospecific SNA lectin binding in purified total IgG (tIgG) in donors and cancer patients, indicating that HAbs are higher sialylated. The avidity of anti-TF IgG in tIgG samples was lower in cancer patients ( The Thomsen-Friedenreich disaccharide Galrcinomas  includinrcinomas  and is aTF, CD 17 is overecroflora .The presence of naturally occurring TF-specific autoantibodies of different subclasses in cancer is a well-documented fact –12. More Sambucus nigra lectin (SNA) than in controls [There is evidence that an appreciable amount of Abs is present in the circulation in a bound form, which phenomenon is called “hidden antibodies” (HAbs), and remains undetectable by conventional serologic methods , 18. No controls . Moreovecontrols . These fαGal IgG, an appreciable amount of natural anti-TF IgG Abs is present in the circulation in a hidden form in both cancer patients and controls. HAbs showed a decreased level in cancer and exhibited a distinct SNA lectin reactivity, avidity, and relation to survival of gastric cancer patients indicating that TF-specific HAbs represent a particular subset of anti-TF IgG Abs which deserves further study to specify their clinical importance.In the present study we show that, in contrast to xenogeneic naturally occurring anti-n = 28) and patients with histologically verified gastric carcinoma (n = 41)  . The inv3), and stored at +4°C until being tested. About 8.5 mg of IgG was obtained from 1 mL of serum applied onto the Protein G Sepharose column. To obtain the IgG-depleted serum we used the same method on the Protein G HP Spin Trap column, except the serum volume applied to the Protein G column was three times lower, and the complete depletion of IgG was controlled using the Easy-Titer IgG Assay Kit .The purification of serum total IgG (tIgG) was performed on the Protein G HP Spin Trap column as described by the manufacturer . The tIgG samples were eluted at pH 2.5, immediately neutralized, dialyzed against phosphate buffered solution  as described elsewhere [α- or αGal-polyacrylamide conjugate  in carbonate buffer, pH 9.6, 5 μg per well. After overnight incubation at +4°C, triple washing, and blocking with Superblock solution  for 15 min at 25°C, the serum or purified IgG samples diluted to 1 : 25 in PBS-0.05% Tween (Tw) were applied for 1.5 hr at 25°C. The concentration of IgG in serum and tIgG samples was measured by the Easy-Titer IgG Assay Kit  and the IgG concentration in the tIgG probe adjusted to that in serum. After subsequent washing with PBS-Tw, the bound anti-TF or anti-αGal IgG was detected with alkaline phosphatase conjugated goat anti-human IgG  and p-nitrophenylphosphate disodium hexahydrate . The absorbance values were read at 405 nm .The anti-TF and anti-lsewhere , 21 withThe IgG concentration in tIgG samples was adjusted to that in serum as described above and the level of anti-TF IgG Ab in serum and tIgG samples was measured by ELISA. The level of hidden IgG antibodies (HAbs) was calculated as the difference between the levels of anti-TF IgG  in tIgG and serum.n = 5) and controls (n = 5) were incubated with an equal volume of the autologic IgG-depleted serum diluted from 1 : 10 to 1 : 100, or bovine serum albumin  for 15 min at 25°C, and the HAb levels  after incubation with PBS-BSA or after addition of IgG-depleted serum dilutions were determined as described above and presented in To evaluate the impact of IgG-depleted serum the purified tIgG samples of cancer patients ( Sambucus nigra agglutinin (SNA) and mannose-specific concanavalin A (ConA) to the absorbed serum anti-TF Abs  or anti-TF IgG from tIgG samples was measured as described elsewhere [2, pH 7.5, and biotinylated ConA  in the ConA binding buffer  were both applied at a concentration of 5 μg/mL each, for 1.5 h at 25°C. The bound lectins were detected with a streptavidin-alkaline phosphatase conjugate  and p-nitrophenylphosphate . The optical density value of control wells (no sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analyzed in duplicate.The lectin reactivity of the TF-specific IgG was measured by ELISA in a similar way, except that the binding of neuraminic acid-  specificlsewhere , 21. Bio4SCN). This concentration of the chaotrope was selected in the preliminary titration experiments. Under the conditions used, about 60% of serum IgG antibodies of controls were detached after treatment with 1.25 M thiocyanate. From each sample, a series with and without thiocyanate treatment were made.The assay is based on the dissociation of Ab-Ag complexes by 1.25 M ammonium thiocyanate . After incubation for 1.5 hr at 25°C the wells were exposed to either PBS-0.05% Tw or NH4SCN solution (1.25 mol/L) for 15 min at +25°C. After triple washing, the wells were incubated with alkaline phosphatase-labeled anti-human IgG for 60 min at 37°C. The bound antibodies were detected with alkaline phosphatase conjugated goat anti-human IgG  and p-nitrophenylphosphate . The results were expressed as a relative avidity index (AI) representing the percentage of reactivity remaining in the thiocyanate treated well.Purified total IgG samples with an adjusted IgG concentration and serum samples diluted to 1 : 25 were added to the wells coated with TF-PAA or  U test for unpaired data or Student's t-test, where appropriate, and the Pearson two-tailed correlation. A P ≤ 0.05 value was considered statistically significant. Survival analysis was carried out by the Kaplan-Meier method using the Estonian Cancer Registry database. The group median was used as a cut-off limit. The differences between cancer patients and controls in Ab levels and the avidity were evaluated for the diagnostic accuracy for cancer by the receiver operator characteristic (ROC) curve analysis. All calculations were performed using the GraphPad Prism 5 and SPSS 15.0 software.Comparisons between the groups were performed using the nonparametric Mann–WhitneyP = 0.047 and 0.011, resp.) in cancer patients compared to controls. The OD values were significantly higher in purified tIgG than in serum samples in donors (P < 0.0001) and cancer patients (P < 0.0001)  . Only onobserved .P = 0.04). This decrease was mostly associated with stage 3 patients unlike controls (P = 0.005) (P = 0.01) compared with males . In contrast to anti-TF IgG, the level of hidden anti-αGal IgG Abs was very low and showed no difference between controls and patients . In eight of 20 controls and seven of 16 patients the level of anti-αGal IgG in tIgG was even lower than in serum samples.The level of hidden antibodies (HAb) defined as the difference between the levels in tIgG and serum was lower in cancer patients (= 0.005)  and relaαGal Abs where the HAbs level was very low.It is notable that after incubation of purified tIgG with IgG-depleted autologic serum the level of anti-TF IgG HAbs decreased dramatically and dose-dependently nearly down to the level in the serum already at the 1 : 10 dilution of the IgG-depleted serum . The impαGal hidden IgG was demonstrated in both gastric cancer patients and controls. A decreased level of anti-TF IgG HAb was observed in gastric cancer patients, especially in stage 3 females. It appears that the autologic IgG-depleted serum contains ligands that react with anti-TF IgG Abs, making them undetectable in the serum.Thus, rather a high level of hidden anti-TF IgG Abs but not anti-P < 0.0001) in patients and controls , whereas no difference between patients and controls was found for tIgG samples .The levels of SNA and ConA binding to anti-TF Abs were much higher in serum than in total IgG samples  and, to a lesser extent, in cancer patients  , while no difference in cancer patients was observed (P = 0.80) (P = 0.025). The avidity of free serum anti-TF IgG was similar in patients and controls (P = 0.47).In donors, the avidity of anti-TF IgG Abs in purified tIgG was found to be higher than that in serum ( = 0.80) . Moreover = −0.75 and r = −0.63 in controls and cancer patients, respectively (P < 0.0001) , while no correlation between the HAbs level and IgG avidity was found in patients with cancer   (Changes in the anti-TF IgG HAb level in the whole cancer group showed rather low accuracy (area under the ROC curve (AUC) = 0.61, 95% CI 0.47–0.75) as analyzed by ROC curve analysis. The sensitivity increased in stage 3 patients: AUC = 0.69 (95% CI 0.54–0.84) . Interes 0.0094) , compareP = 0.052). A better discrimination of patients and controls was obtained by using the ratio: the avidity of anti-TF IgG in tIgG samples/the avidity of IgG in serum samples  especially in stage 3 males  , whereas no relation of this level in purified tIgG samples to survival was found , P = 0.98, Figures P = 0.07). This was mostly on account of stage 3 patients (HR = 0.34 (0.09–1.26), P = 0.10). No association of the avidity of serum anti-TF IgG Abs with survival (HR = 1.02 (0.43–2.41), P = 0.95) was established ,  Figures . Survivaablished .In summary, the level of HAbs and the avidity of anti-TF IgG showed a moderate stage- and gender-dependent diagnostic accuracy. A better outcome was observed in patients with a high level of TF-specific IgG in serum (stage 3), in those with a high level of SNA binding to serum anti-TF Abs , and in patients with a low avidity of anti-TF IgG antibodies in purified tIgG.In this study, we focused on the anti-TF IgG HAb for several reasons. First, since the Fab fragment is not involved in the binding of IgG to PtG, the tIgG preparations are enriched in immune complexes, while the use of glycan- (TF-) specific sorbents for anti-TF Ab purification from the serum will exclude such a possibility due to the involvement of Fab fragments in the purification process. Second, the higher avidity of IgG allows the preferential binding of TF-positive ligands despite the possible interference of the other Ig isotypes. Third, using the Protein G it is much easier to purify IgG without the other Ig isotypes admixture. Fourth, the changes in the anti-TF IgG level showed a significant association with cancer progression and patients survival , 13 thusαGal HAbs or a very low amount of them was found in the same samples. Differently from controls a decreased level of anti-TF IgG HAbs was found in cancer patients (P = 0.005 for stage 3 patients).The results obtained in this work show that anti-TF IgG HAbs are present in the blood of both cancer patients and healthy controls. In contrast, no anti-αGal-specific HAbs in the same purified tIgG samples strongly suggests that the purification of IgG per se is not a sufficient reason to explain the appearance of IgG HAbs. Moreover, the inhibition of the reactivity of hidden Abs by addition of IgG-depleted serum implies the role of serum-derived factors such as TF-positive or cross-reactive ligands that remain in the IgG-depleted serum and react with anti-TF IgG Abs again, making them HAbs. This restoration phenomenon .It has been shown that the in vitro exposure of human IgG to protein-destabilizing chemical or physical factors results in the exposure of IgG “hidden” reactivity . Howeverenomenon  was abseγ receptor interactions and thus the Fc-mediated effector mechanisms [Immunoglobulins (Igs) are glycosylated molecules which display a set of glycoforms that differ in number, type, and site of oligosaccharide attachment . The N-gchanisms , 28. Agachanisms –31 thougchanisms . It has chanisms . It is nchanisms , 35.There is evidence that the aberrantly glycosylated serum IgG may be either of tumor origin, or accumulated in tumor tissue . The higP = 0.03) (The comparison of lectin binding patterns of anti-TF Abs in serum and tIgG shows that the main targets for binding SNA and ConA lectin in serum are TF-specific IgM and/or IgA. The SNA binding to free anti-TF Abs in serum was significantly higher in cancer patients ( = 0.03)  whereas It is known that the total IgG purified on PtG sorbents is enriched by immune complexes and contains a lot of so-called “hitchhiker” ligands , 43. We Contrary to blood donors, cancer patients demonstrated a lower avidity of anti-TF IgG in tIgG samples while no differences in the avidity of free serum TF-specific Abs between the two groups were observed. Since the tIgG contains both free Abs and HAbs, this decrease is obviously related to the HAbs that display a lower avidity in cancer.The lower level of HAbs showed a rather good diagnostic accuracy for cancer in stage 3 females (AUC = 0.78) compared to males (AUC = 0.55). In contrast, the ratio of anti-TF IgG avidity in tIgG to the avidity of IgG in serum revealed a better diagnostic value in males (AUC = 0.784) than in females (AUC = 0.629) . Since tr = −0.54, P = 0.003) but not in cancer patients  , whereas no connection with the SNA reactivity of anti-TF IgG in tIgG (HR = 1.01 (0.4–2.5), P = 0.98) was demonstrated. We have shown recently that the higher avidity of SNA-reactive TF-specific Abs in the serum of cancer patients was not related to the IgG isotype [It has been reported that the Fc glycan of adaptive IgG Abs is lower sialylated , though  isotype . In the It is to be noted that despite the TF expression on tumor cells there is no evidence that any adaptive humoral immune response to TF occurs in the tumor host. Instead, a decrease in the level of serum TF-specific Abs is usually observed –11, 13. In conclusion, TF-specific HAbs represent a particular subset of anti-TF IgG Abs that differ from free anti-TF IgG Abs in SNA reactivity, avidity, diagnostic potential, and relation to survival. Thus it appears that free serum anti-TF natural Abs in the circulation are only the “tip of the iceberg.” The hidden anti-TF antibody reactivity emerging in purified IgG suggests that serological testing with whole serum does not reflect the whole picture, and the HAbs analysis could tell us more about the other players and targets involved. Given that there are still no reliable biomarkers for gastric cancer, the changes in the level of anti-TF hidden IgG antibodies and their avidity found in this study merit further study. Whether the tumor-specific ligands are involved in the HAbs formation in patients with cancer remains to be clarified. The functional activity and clinically important effects of anti-TF Abs may apply to only a particular subset/glycoform of TF-specific Abs which contributes to cancer and its progression. Further characterization of TF-specific antibody subsets and potential ligands involved in the HAbs formation in health and disease may extend the concept of natural autoantibody signatures and improve the clinical utility of antibody-based diagnostic and predictive biomarkers.


Most cancer studies focus on exploring non-invasive biomarkers for cancer detection. In the present study, we sought to investigate the expression level of microRNA-21 (miR-21), as a potential diagnostic marker, in serum and stool samples from 40 patients with colorectal cancer (CRC) and 40 healthy controls.Quantitative real-time RT-PCR was applied to determine the relative expression level of miR-21 in serum and stool. At the same time, the sensitivity and specificity of this marker was evaluated by receiver operating characteristic (ROC) curve analysis.P<0.05, 95% CI: 5.774-34.045) and 10.0  times in CRC patients, respectively, when compared to the control group. The sensitivity and specificity of miR-21 was found to be 86.05% and 72.97%, respectively (an area under the ROC curve [AUC] of 0.783). The stool miR-21 level in CRC patients was much higher than that in the healthy controls, showing a sensitivity of 86.05% and a specificity of 81.08% (AUC: 0.829). The expression level of miR-21 in stool was able to significantly distinguish CRC tumor, node, metastasis stages III-IV from stages I-II, with a sensitivity and specificity of 88.1% and 81.6%, respectively (AUC: 0.872).miR-21 expression levels of serum and stool were up-regulated 12.1 (The results of this study indicated that miR-21 expression levels in serum and stool can be considered as a potential diagnostic biomarker for the diagnosis of CRC patients. However, more studies are required to confirm the validity of miR-21 as a valuable non-invasive diagnostic tool for CRC. Nowadays, non-invasive biomarkers are being widely explored as a reliable tool for cancer diagnosis. Colorectal cancer (CRC) is the second leading cause of cancer death in the world,2. TypicCRC is a multi-stage process specified by genetic and epigenetic variations that affect the main cellular pathways involved in growth and development. TherefoMiRNA-21 is an oncogenic miRNA that regulate the expression of certain target genes such as programmed cell death 4, phosphatase and tensin homologue and tropomyosin 1. Recent Totally, 40 CRC patients and 40 healthy controls were participated in this study . Blood aPrior to surgical tumor resection and seven days after colonoscopy, whole-blood samples were collected from each participant into 5-ml RNase-free tubes. The serum was then separated and stored at -80°C until use. Stool samples were collected simultaneously from the both groups as the whole blood samples, immediately flash frozen in liquid nitrogen, and stored at -80°C. On the sampling day, the following basic demographic and clinical information was collected from all participants: cancer stage at diagnosis, the histological type and the grade of the tumor. The staging of tumors was determined according to the American Joint Committee on Cancer TNM staging system,25. It sMore importantly, miRNeasy procedures minimize the possibility of contamination with salts or phenol, which could interfere with later analyses. The total amount of serum samples used for this kit was 200 µl. Total miRNAs were extracted according to the manufacturer’s instructions. The concentration and purity of the extracted miRNAs were assayed using the Eppendorf BioPhotometer .-ΔΔCT method [ΔΔCT=ΔCT (a miRNA of interest)-ΔCT ] was used to analyze the relative expression of this miRNA) was maximum. The optimal cut-off values were used to calculate the sensitivity and specificity as well as positive and negative predictive values. The cut-off value estimated for serum and stool miR-21 levels was 1.1 and 3.05, respectively.As shown in Molecular techniques, especially gene expression profiling, are commonly used to improve CRC classification, elucidate patients’ prognosis and predict response to therapy,28. EarlTo date, several interesting miRNAs have been candidated as CRC molecular biomarkers; however, it is difficult to draw a definite conclusion-35. The et al.[Previous studies have indicated that the miR-21 expression level is increased in different TNM stages of CRC, from the early to later stages,33. In tet al.3, it turet al.. In mostet al.,30-34. Set al., we atteIn summary, serum and stool miR-21 expression levels seem to be a potential diagnostic molecular biomarker for CRC patients. However, well-designed studies with larger sample sizes are recommended to further investigate the role of miR-21 in CRC diagnosis.


Background. Ranibizumab is approved for treatment of macular edema in eyes with retinal vein occlusion (RVO). Some eyes show low-response to treatment with regard to visual acuity gain (VA) and OCT central retinal thickness (CRT) reduction. The goal of this study was to quantify the percentage of low-responders. Methods. Treatment of naïve eyes with macular edema secondary to RVO was included and monthly VA and CRT were analyzed. Four weeks after the loading phase, and at the end of the study, eyes were grouped into low- and good responders based on predefined criteria. The responder and low-responder groups were then compared at various time points. Results. Forty-three eyes were included. Regarding VA, 27.9% were low-responders after the loading phase and 30.2% at the end of the study. For CRT, 34.9% were low-responders after the loading phase versus 27.9% at the end of the study. 75% of patients that were VA low-responders and 73.3% of CRT low-responders after loading phase remained low-responders at the end of the study. Conclusion. Approximately 30% of patients showed low response to ranibizumab after the loading phase and after 1 year of treatment. Two-thirds of patients that were low-responders after the loading phase remained low-responders after 1 year. Central retinal vein occlusion (CRVO) is characterized by blockage of the major outflow vessel of the retina, resulting in retinal hemorrhages, exudates, and macular edema . In branIntravitreal injection of ranibizumab , a Fab fragment that binds all isoforms of VEGF-A, has been shown to markedly reduce macular edema. Two large, multicenter, double-masked trials—the Ranibizumab for the Treatment of Macular Edema following Branch Retinal Vein Occlusion Study (BRAVO)  and the NCT00379795) was designed to obtain additional information about the long-term efficacy of ranibizumab treatment in two patient cohorts. Cohort 1 included patients with neovascular age-related macular degeneration and will not be discussed further. Cohort 2 included patients with macular edema after retinal vein occlusion that had completed the BRAVO or CRUISE studies [The HORIZON trial . At baseline and each following month best-corrected ETDRS visual acuity (VA) and OCT central retinal thickness  were measured. Final VA and CRT were noted at month 12 or in case of an injection, 4 weeks after the last injection. For OCT retinal thickness measurements, Spectralis OCT volume scans  were used. The follow-up function was used for repeated measurements. Patients were categorized into a treatment responder and a low-responder group based on VA and CRT change. The first cut-off was made at month 4, four weeks after the loading phase of three consecutive, monthly ranibizumab injections. The second cut-off was made at the end of the observation period. Responder and low-responder groups were then compared. Eyes were characterized as low-responders if VA did not increase >6 letters from baseline, which represents less than 50% of the expected VA gain extrapolated from the BRAVO and CRUISE studies [ letters.t-test. Differences between good responders and low-responders were tested with the Chi-Square test. p values <0.05 were considered statistically significant. All values are presented as mean ± SD.GraphPad Prism software (version 5.02) was used for statistical analysis. Group means of low-responders and responders in VA and CRT at different time points were tested with Student's The study followed the tenants of the Declaration of Helsinki and was approved by the Cantonal Ethics Committee Berne.μm. Final VA increased to 64.0 ± 17.2 letters and CRT decreased to 328.8 ± 139.3 μm. Differences in VA and CRT change were significant (p < 0.0001). 176 charts of patients with macular edema due to CRVO or BRVO were identified. 43 eyes were included in the analysis . All other eyes have been excluded because either they were not treatment naïve or treatment was switched to other intravitreal therapy during the first year. Mean baseline VA was 47.4 ± 17.3 letters. Mean baseline CRT was 588.7 ± 193.9 p = 0.0002) and CRT (p < 0.0001). The corresponding bar plots are shown in p = 0.0067). VA low-responders after the loading phase started with a significantly better baseline VA  compared to treatment responders. Final visual acuity was significantly lower in the VA low-responder group at the end of study . In addition, low-responders at the end of study already showed significantly lower VA values after the loading phase . Further, VA low-responders after the loading phase had significantly lower VA values compared to VA responders after the loading phase . At the end of study, VA low-responders after the loading phase and at the end of study showed significantly higher CRT values at the end of study .Group comparison revealed that VA low-responders after the loading phase required significantly more injections within the first year of treatment .Finally, VA low-responders after the loading phase and at the end of study showed significantly higher CRT values after the loading phase . Two low-responder patients showed ischemic areas with capillary dropout in the macula  during fluorescein angiography, versus 4 patients in the responder group .μm versus 268.9 ± 53.1 μm; p = 0.0001) and at the end of study . If patients were CRT low-responders already after the loading phase, mean CRT values were also significantly higher at the end of study . In addition, CRT low-responders after the loading phase showed significantly lower VA values after the loading phase . CRT low-responders at the end of study already showed significantly lower VA values after the loading phase .CRT low-responder received a mean number of 9.2 injections versus 8.8 injections in the nonresponders group. In addition, CRT low-responders showed significantly higher CRT values compared to responders after the loading phase , versus 4 patients in the responder group .No other significant differences between the low-responder and responder groups were found for age, baseline VA, or baseline CRT.p = 0.027).Low-response to antiVEGF treatment is frequently observed in clinical routine. Just recently, Guber et al. described certain clinical risk factors for low response to ranibizumab in exsudative age-related macular degeneration . For macNevertheless, a certain percentage of retinal vein occlusion patients show low-response to anti-VEGF treatment. Noma et al. published two studies measuring VEGF and Interleukin 6 (IL6) levels in the vitreous of eyes with CRVO and BRVO , 9. MeanRecently, new drugs became available for treating macular edema in eyes with retinal vein occlusion. Ozurdex™, a biodegradable dexamethasone implant has been shown to be effective in reducing macular edema and improving patient's vision in a prospective randomized trial . AlthougThis study has some limitations. First, this was a retrospective study design with a relatively small study population. Second, patients that were switched to other intravitreal treatments during the observational period have been excluded in order to assess solely the effect of ranibizumab on VA and CRT. Therefore, the study population is biased and the rate of low-response to treatment might be underestimated. Third, no VEGF levels were assessed in our study group. In addition, other morphological changes or baseline values such as the presence of retinal hemorrhages, hard exudates, or concomitant diseases have not been analyzed.In conclusion, approximately 30% of patients with macular edema due to retinal vein occlusion showed a low response to ranibizumab treatment after the loading phase and after 1 year of treatment. Two-thirds of patients that were low-responders after the loading phase remained low-responders after 1 year of treatment. Based on our data, a switch of therapy to another intravitreal drug should be considered after the loading phase if a low response is observed. A prospective, randomized comparative trial is needed to find out which therapy switch might actually be beneficial for our patients.


To investigate optic disc hemorrhage (DH)’s clinical implications to subsequent progression of primary open-angle glaucoma (POAG) in cases of mild-to-moderate myopia.Retrospective comparative study.(1) Fifty-nine (59) myopic  POAG patients with DH and (2) 59 age-, AXL-, and mean deviation (MD) of visual field (VF)-matched controls without DH were evaluated over the course of a minimum 3.5 years of follow up. For clear assessment of the effect of DH on progression of glaucoma, the patients selected for inclusion in the study were those with stable IOP  whose IOP-lowering medication was not increased, supplemented or changed during the follow-up period.The patients’ optic disc/retinal nerve fiber layer (RNFL) photographs were independently evaluated by three glaucoma specialists for structural progression of glaucoma. Event-based analysis with Guided Progression Analysis (GPA) software was used to determine their functional progression. The durations of structural and functional progression were compared by Kaplan-Meier life survival analyses.Optic disc/RNFL progression and VF progression.The mean follow-up periods between the DH and non-DH groups were not significantly different: in the DH group, 5.6±2.7 years; in the non-DH group, 5.4±2.6 years (P = 0.588). In the DH group, 30 (50.8%) of 59 eyes manifested optic disc/RNFL deterioration; in the non-DH group, however, only 17 (28.8%) of 59 eyes showed structural progression. For the DH group, the cumulative probability of structural glaucoma progression was significantly greater than for the non-DH group . Interestingly, the two groups did not significantly differ in the cumulative probability of functional progression : in the DH group, VF progression was observed in 14 eyes (23.7%); in the non-DH group, in 12 eyes (20.3%).DH was associated with a greater probability of structural progression in medically well-controlled-IOP POAG eyes with mild-to-moderate myopia. However, the relevance of DH to VF progression was not clear over the course of the average 5.5-year duration of the study. Optic disc hemorrhage (DH) is one of the most important risk factors with respect to glaucoma progression –4. ThereThe pathophysiology of DH has not yet been fully elucidated, despite its strong association with glaucoma progression. According to the mechanical hypothesis, DH is the consequence of microvascular disruption resulting from structural alteration of the lamina cribrosa (LC) ,10; accoIn myopic eyes, axial elongation of the eyeball is accompanied by structural changes to the optic disc and/or peripapillary retina ,14. TiltNo previous research has clearly demonstrated the effect of DH on subsequent progression in glaucoma eyes with myopia. Thus, we carried out the present study to evaluate myopic patients’ clinical-DH implications for glaucoma progression, specifically by comparing myopic primary open-angle glaucoma (POAG) eyes with and without DH.This study was approved by the Seoul National University Hospital Institutional Review Board and adhered to the tenets of the Declaration of Helsinki.Patients who had been examined at Seoul National University Hospital’s Glaucoma Clinic from January 2005 to May 2013 and followed up on at 6-month intervals for a minimum of 3.5 years were consecutively included in this study after a retrospective medical-record review. All underwent a complete ophthalmic examination: visual acuity assessment, refraction, slit-lamp biomicroscopy, gonioscopy, Goldmann applanation tonometry , and dilated-funduscopic examination. Additionally, they were subjected to the following: central corneal thickness measurement , AXL measurement , digital color stereo disc photography (SDP), red-free RNFL photography, optic nerve head imaging by Cirrus spectral domain-optic coherence tomography SD-OCT; Carl Zeiss Meditec, Dublin, CA), and Humphrey Visual Field central 30–2 threshold tests .Upon enrollment in the study the eligible participants had POAG, mild-to-moderate myopia (AXL between 24.0 mm or longer and less than 26.5 mm) ,19, and Patients were excluded from further analysis any of the following reasons: history of intraocular surgery (exception: uncomplicated cataract); any posterior-pole lesions possibly affecting VF examination results; current usage of oral antiplatelet and/or anticoagulation medication; associated systemic disease(s) other than hypertension and well-controlled diabetes mellitus. All of the included patients’ eyes had well-controlled IOP: that is, all having received one or more topical glaucoma medications, and all showed at least 20% IOP decreases from the baseline. For accurate assessment of DH’s effect on glaucoma progression, only stable-IOP patients for whom IOP-lowering medication was not increased, supplemented or changed until a definite VF progression was found were included in this study. As for those without clinically detected DH, age-, AXL- and MD-of-VF-matched eyes were selected for inclusion in the non-DH group.β-zone parapapillary atrophy (PPA) area  and the clinical disc margin were plotted by using a mouse-driven cursor to trace the disc and PPA margins directly onto the SDP image. Then the pixel areas of the β-zone PPA and clinical disc were obtained with the ImageJ 1.40 software . The β -zone PPA area was calculated from the ratio of PPA pixel area to the disc pixel area as previously described method [The optic disc area was calculated in consideration of the magnification factors related to the SD-OCT camera and the eye by substituting the subject's AXL as previous study . The β-zd method .During the follow up, all of the patients had their SDPs repeated every 6 months at the minimum. These images were retrospectively and independently evaluated for DH by three glaucoma specialists  blinded to the patients’ clinical information. For any of the following reasons, DH was considered to be unrelated to glaucoma: (1) the disc was swollen, or otherwise obviously abnormal, due to non-glaucomatous optic neuropathy; (2) there were multiple proximitous retinal hemorrhages suggestive of either diabetic retinopathy or retinal vascular abnormality; (3) there was acute posterior vitreous detachment possibly causative of DH. In bilateral DH cases meeting all of the inclusion criteria, just one eye was selected randomly. Only patients who had developed DH during interval testing were enrolled in the study.Three experienced graders  blind to the patients’ clinical and VF information reviewed each patient’s SDPs and RNFL photographs. They then determined, based on the initial photograph, structural progression’s presence or absence. Optic disc progression was considered to be the extent of neuroretinal rim thinning. RNFL progression was defined as widening, deepening, or a newly apparent RNFL defect. If eyes had diffuse atrophy or invisible RNFL, and if evaluation of RNFL photography, consequently, was difficult, progression was determined solely based on optic disc evaluation. In cases of eyes with severe optic disc tilt and/or torsion rendering neuroretinal rim evaluation unreliable, confirmation of progression was made based only on RNFL photography assessment. Each glaucomatous eye was classified by each of the graders as either stable or progressing.If the opinions of the three examiners on DH discrimination or structural progression differed, post-discussion consensus was reached. If no consensus could be reached, the pertinent eye was excluded from subsequent analysis.The patients’ VF test results were evaluated to assess functional glaucoma progression. The first 1 to 2 VF results were excluded to minimize the learning effects, and any unreliable results  were also excluded. VF progression was determined by event-based analysis using the Humphrey Field Analyzer with Guided Progression Analysis (GPA) software. VF progression was defined as a significant decrease from the baseline pattern deviation at three or more of the same test points on two or three consecutive VF tests. The GPA software classifies VF progression as either ‘‘possible progression” or ‘‘likely progression.” In the current study, only ‘‘likely progression” was considered to be VF progression. A glaucoma specialist (YKK) reviewed all the patients’ VF results to ensure that there was no artefactual software analysis included.t-test. The categorical data, meanwhile, were analyzed by χ2 test. The cumulative risk ratios of structural and functional progression were compared between the groups by Kaplan-Meier survival analysis and log rank test. The initial progression detection was regarded as the endpoint in the survival analysis. The end of follow up was deemed to be the point at which patients without progression were censored. The statistical analysis was performed using the SPSS statistical package . A P value less than 0.05 was considered to be statistically significant.The DH and non-DH groups’ demographic and clinical characteristics were compared. The comparison of normally distributed data was performed using the independent Student’s A total of 1048 myopic glaucoma patients’ medical records were initially reviewed for the purposes of the present study, and 80 DH eyes that met the inclusion and exclusion criteria were selected. One (1) case was excluded from subsequent analysis because the opinions of the three examiners on structural progression differed, and no consensus could be reached. Among the remaining subjects, 3 eyes were excluded from further analysis due to myopia-related retinopathy diagnosed over the course of follow-up. Seventeen (17) patients who had received post-DH treatment reinforcement immediately after the occurrence of DH also were excluded.The DH group finally included 59 myopic glaucoma patients. The mean follow-up period after initial DH was 5.6 ± 2.7 years. 13 eyes (22.0%) of DH group showed multiple DHs during the follow-up periods. 8 eyes had 2 episodes of DH, 5 eyes had more than 2 episodes of DH. For the purposes of comparative analysis, age-, AXL- and baseline MD-of-VF-matched controls (59 patients) who did not incur DH during the follow-up period were included in the non-DH group. Their mean follow-up period (5.4 ± 2.6 years) did not significantly differ from those of the DH group .In the DH group (59 eyes), progression was observed in 30 eyes (50.8%) by optic disc/RNFL photographic assessment, in 14 eyes (23.7%) by VF analysis, and in 38 eyes (64.4%) by either optic disc/RNFL or VF examination. The mean intervals between DH and structural/VF progression were 2.7 ± 1.1 and 6.1 ± 2.2 years, respectively.In the non-DH group, 17 eyes (28.8%) showed progression by optic disc/RNFL photographic assessment, 12 eyes (20.3%) by VF analysis, and 19 eyes (32.2%) by either optic disc/RNFL or VF exams. The mean intervals between the baseline and structural/VF progression were 3.5 ± 2.33 and 5.6 ± 2.45 years, respectively.The cumulative probability of structural progression was significantly greater for the DH group than that for the non-DH group  probability for myopic POAG eyes with DH than for eyes without DH: of the patients who showed post-DH structural progression in this study, 88% of the eyes manifested DH-adjacent RNFL defect progression. The association between DH and progressive glaucomatous changes already are well documented –4. NittaInterestingly, we were unable to show statistically significant differences between our two groups (DH and non-DH) in VF progression. That is, the relevance of DH to VF progression was not clear over the course of the average 5.5-year duration of the study. In glaucoma, it has been well known that structural damage precedes functional deterioration –28. PrevFor clear assessment of the effect of DH on progression of glaucoma, the patients selected for inclusion in the study were those with stable IOP  ,34 whoseThe present study’s findings should be interpreted with mindfulness of its limitations. First, we cannot exclude the possibility of selection artifacts. It is possible that patients whose DH was missed were included in the non-DH group, because whereas DH has been reported to last for only 2 to 6 months, disc photographs were recorded at 6-month intervals. However, misclassification of patients with DH to non-DH group would bias the results toward the null. So statistically significant difference that found between the groups in this study might imply even stronger association. Certainly, the frequency of follow-up would impact DH- and progression-detection rates. Although the average numbers of SDP/RNFL photographs and VF examinations were equivalent in the groups with and without DH, there is a possibility that patients at greater risk had been followed up more frequently. Second, glaucomatous progression for each patient was evaluated only after the development of DH, and deterioration before the event was not assessed. Such analysis would require that all patients had at least 5 VF tests before DH detection and sufficient follow up duration, which was not available for most of the enrolled subjects. De Moraes et al. reported that spatially consistent VF progression may occur before DH detection . Third, In conclusion, DH was associated with a greater probability of optic nerve head/RNFL progression in POAG eyes with mild-to-moderate myopia. Over the course of the 5.5-year follow up, however, there were no significant differences in VF-deterioration probability between myopic POAG eyes with and without DH. Determination of whether post-DH treatment reinforcement can be of any significant benefit to myopic POAG eyes with stable IOP will have to await further study.S1 Table(XLSX)Click here for additional data file.


The majority of the hydroperoxides were detected only in the HDL fraction, not in the LDL/VLDL. Further detailed analysis of HDLs by size-exclusion high-performance liquid chromatography revealed that the hydroperoxide-containing molecules were small-sized HDLs. Because HDL was shown to be the principal vehicle for the plasma hydroperoxides, this Fe-ROMs test is a beneficial method for the assessment of oxidized-HDL levels. Indeed, Fe-ROMs levels were strongly associated with the levels of oxidized HDL, which were determined by performing the malondialdehyde-modified HDL enzyme immunoassay. In conclusion, the Fe-ROMs test using plasma itself or the HDL fraction after dextran sulfate-Mg2+ precipitation is useful to assess the functionality of HDL, because the oxidation of HDL impairs its antiatherogenic capacity.Reactive oxygen species (ROS) are involved in the initiation and progression of atherosclerosis. ROS-derived hydroperoxides, as an indicator of ROS production, have been measured by using the diacron reactive oxygen metabolites (d-ROMs) test, which requires iron-containing transferrin in the reaction mixture. In this study we developed a modified d-ROMs test, termed the Fe-ROMs test, where iron ions were exogenously added to the reaction mixture. This modification is expected to exclude the assay variation that comes from different blood iron levels in individuals. In addition, this Fe-ROMs test was helpful for determining the class of plasma lipoproteins that are hydroperoxidized. Low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and high-density lipoprotein (HDL) were purified by use of an LDL/VLDL purification kit and the dextran sulfate-Mg They are generated by the consumption of O2 and thought to cause deleterious oxidative damage to virtually all molecules. These reactive species are not necessarily a threat to the body under normal physiological conditions [Oxidative stress can be caused by excess reactive oxygen species (ROS) and reactive nitrogen species. ROS include superoxide , and 4-hydroxy-2-trans-hexenal (HHE) [S)-2-amino-6--hexanoic acid result from the covalent chemical adduction of MDA with protein-bound lysine residues [ROS oxidize various biological macromolecules such as proteins, lipids, and DNA, thus causing structural and functional changes in them. Among these molecules, lipids in membranes and lipoproteins are prominent oxidation targets. Lipid oxidation generates hydroperoxides, which subsequently undergo fragmentation to produce a broad range of reactive intermediates such as the arachidonic acid-derived prostaglandin F2α isomer isoprostanes (IsoPs), malondialdehyde (MDA), 4-hydroxy-2-al (HHE) ,6,7,8,9.residues ,11. Manyresidues . In addiresidues ,14.p-phenylene diamine hydrochloride. This test is a simple and an easy way of detecting hydroperoxides, but there are some factors limiting its widespread use: (1) a high-priced machine  or specifically designed machine  is required; (2) the chemical entities measured are still elusive; and (3) the antioxidant substances and blood levels of metal ions, such as iron, may affect the measurement accuracy. Therefore, simpler and more reliable methods for analysis of hydroperoxides are required.The diacron reactive oxygen metabolites (d-ROMs) test can quantify the oxidative stress status by measuring hydroperoxides and has been introduced to analyze their levels in serum or plasma. This test is based on the principle that iron ions released from serum proteins under acidic conditions (pH 4.8) stimulate the conversion of hydroperoxides to alkoxyl and peroxyl radicals, which subsequently react with the chromogen diethyl When blood samples are used for the determination of hydroperoxides, lipoproteins are the most likely entities measured by the d-ROMs test. Oxidatively modified low-density lipoprotein (oxLDL) is one form of modified LDL that has been demonstrated to be present in vivo , specifiIn this study, we developed a modified d-ROMs test that could serve as a useful basis for evaluating the oxidative status of plasma. Furthermore, we studied whether our d-ROMs test would be applicable for the assessment of oxLDL and oxidized-HDL (oxHDL) levels of human plasma. This work proves the applicability of our modified test for evaluating the level of oxHDL.N,N′-diethyl-p-phenylenediamine and an aliquot of plasma were added into each of the 96 wells in a micro-plate and incubated in the presence or absence of 100 µM Fe2+ for 10 min at 37 °C. As shown in 2+, and its rate of increase during the 120 to 300 s was 37.005 mOD /min in the presence of Fe2+ and 11.868 mOD/min in the absence of it. This Fe effect was also observed in the presence of Fe3+ , although the ratios of Fe-ROMs levels to d-ROMs levels tended to become higher as the d-ROMs levels increased. The correlation between both levels was also strong in five healthy subjects . For this experiment, we chose subjects with acute febrile disease because they show a wide range of d-ROMs levels from normal to high. Indeed, their hydroperoxides levels ranged from 205 to 430 Carratelli units (U.CARR) . The levThe Fe-ROMs test has some advantages over the original one. In the original test, both hydroperoxides and iron ion are supplied by plasma; but in the Fe-ROMs test, iron ions are supplied exogenously in the reaction mixture. Therefore, the Fe-ROMs test allowed us to fractionate hydroperoxide-containing molecules from plasma without paying attention to the presence of iron-supplying molecules. We first separated plasma into filtrate and residual fluid by using an Amiconultra-0.5 (100 kDa). The recovery of hydroperoxides in the residual fluid, which was determined by the Fe-ROMs test, was 99.3% (the average recovery of three independent experiments), indicating that most of the hydroperoxidized molecules were included among the macromolecular components.Oxidative stress targets a variety of molecules in plasma. Among them, lipids, especially PUFAs, are most subjected to oxidative stress, leading to lipid peroxidation. Lipoproteins are macromolecular complexes of phospholipids, free cholesterol, cholesterol esters, triglycerides, and apolipoproteins. Because phospholipids, cholesterol esters, and triglycerides contain PUFAs, it is most likely that hydroperoxides are present in plasma lipoproteins. We then isolated the low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) fraction from the plasma by using an LDL/VLDL Purification Kit. A dextran solution was first added to the plasma to selectively precipitate LDL/VLDL from the plasma, and the pellet obtained by centrifugation was used for the subsequent LDL/VLDL purification. We detected less than 3% of hydroperoxides in the purified LDL/VLDL as compared to the amount in the original plasma sample, indicating that the hydroperoxide level in LDL/VLDL was too low to be detected by the Fe-ROMs test. In contrast, all the activity (105%) was detected in the supernatant obtained after centrifugation following the addition of the Dextran solution, indicating that HDL was probably the major carrier of hydroperoxides in plasma.2+, and assayed its activity by performing the Fe-ROMs test. All the activity (104%) was recovered into the supernatant after the dextran sulfate-Mg2+ precipitation procedure, suggesting that the hydroperoxide-containing molecule was HDL. The lipoprotein separation was quite sensitive to the molecular size of the dextran sulfate, the concentrations of dextran sulfates and Mg2+, and the temperature. Consequently, we evaluated the lipoprotein profiles of the plasma and the supernatant after performing the dextran sulfate-Mg2+ precipitation by using gel-permeation high-performance liquid chromatography (GP-HPLC). As shown in We then isolated the HDL from the other lipoproteins with a combination of dextran sulfate and Mgr = 0.924, As described above, almost all the plasma hydroperoxides were detected in the HDL fraction. Because the levels of Fe-ROMs in the plasma were different from subject to subject, it was interesting for us to compare Fe-ROMs levels in the plasma and its dextran sulfate-supernatant in many subjects. We next fractionated the plasma from a healthy subject by using HPLC and defined the molecules that had been hydroperoxidized. As shown in 2+ precipitation procedure and determined their oxidation levels by performing an MDA-HDL enzyme immunoassay and Fe-ROMs test (r = 0.864), although two subjects with similar Fe-ROMs levels (about 20 mOD/min) showed different MDA-HDL levels.The oxidation of HDL causes a reduction in the ability of HDL to promote cholesterol efflux ,22,23. TOMs test . Both leAs shown in Because there is a close association between oxidative stress and vascular diseases, we need reliable biomarkers to assess such stress. In the present study we assessed oxidative stress in human plasma by use of a modified d-ROMs test  where iron ions were exogenously added to the reaction mixture. Our Fe-ROMs test had some advantages over the original d-ROMs test: (1) The Fe-ROMs test required a smaller volume of plasma to measure oxidative stress; (2) it gave us an opportunity to define hydroperoxidized target molecules in the blood; and (3) it could exclude the assay variation that comes from different blood iron levels in individuals.3+-binding transferrin protein in blood samples. The affinity of transferrin for ferric iron (Fe3+) is extremely high under the extracellular environment (approximately pH 7.4), and weakened in the acidic conditions, leading to Fe3+ dissociation from the protein [3+rather than Fe2+ is mainly used in the acidic conditions of the d-ROMs test. Fe3+ increased absorbance at 505 nm dose-dependently, but to a lesser extent than Fe2+. Therefore, the d-ROMs test might not be strongly influenced by different blood iron levels in individuals. However, we recommend adding exogenous iron ions to the reaction mixture, because adding even a small amount of Fe3+  to the reaction mixture changed the absorbance at 505 nm  in blood samples might interact in the assay reaction and influence the results . Recent ron ions . This obROS oxidize not only lipids but also other biological molecules such as proteins and DNA. However, lipids, especially PUFAs, are the ones most targeted by ROS . Lipid oThe imbalance between circulating levels of lipoproteins (LDL and VLDL) relative to those of HDL is associated with atherogenesis. LDL is a major substrate for oxidation at the arterial wall, and the oxidatively modified form of LDL (oxLDL) is more important than native LDL in the process of atherogenesis ,18,36,37Our HPLC analysis revealed that carriers of hydroperoxides were eluted at a position just after the majority of HDLs were eluted and coincidentally with very small HDL d. HDLs aHDL retards atherosclerosis via multiple mechanisms, including reverse cholesterol transport (RCT) ,45, anti2+ precipitation in a secondary assay.This study showed that HDL was the principal carrier of lipid hydroperoxides in human plasma when the Fe-ROMs test was used for the determination of oxidative stress. This is an important finding because oxHDL, which is a risk factor for developing atherosclerosis, can be assayed by the Fe-ROMs test. Enzyme immunoassay kits have been developed for the detection and quantification of oxHDL in plasma or serum. In these assays, antibodies against the secondary oxidation products of HDL, such as MDA-HDL, have been used. However, these kits are not used routinely in a clinical examination setting because they require much labor and are expensive. On the other hand, our Fe-ROMs test is inexpensive and convenient for the determination of oxHDL. Our study indicated that hydroperoxidized HDL can be estimated from Fe-ROMs levels in plasma. However, as demonstrated by Bowry et al. , LDL car4)2(SO4)2, FeCl3, N,N′-diethyl-p-phenylenediamine, dextran sulfate sodium salt , MgCl2, and human serum albumin were purchased from Wako . Tert-butyl hydroperoxide (70% in water) was purchased from TCI . NaCl was from Sigma-Aldrich . HDL Human ELISA (enzyme-linked immunosorbent assay) Kit (ab125961) came from Abcam . A Bolt LDS sample Buffer, Bolt Reducing Agent, and Bolt MOPS SDS Running Buffer were purchased from the Life Technologies Corporation . The Precision Plus Protein Dual Color Standards were from Bio-Rad Laboratories, Inc. . The OH pak SB-804HQ column was obtained from Showa Denko KK .Fe and 10 male patients (age: 15.7 ± 12.1) who had experienced acute febrile disease were briefed about the study protocol and possible risks, signed the informed consent, and voluntarily participated in the present study. The study protocol was approved by the Ethics Committee of Kyoto Prefectural University of Medicine . The patients were included in this study, because they showed a wide range of d-ROMs levels from normal to high. Blood samples were collected from the antecubital vein into sodium heparin-containing blood collection tube for the measurement of reactive oxygen metabolites. The blood samples were centrifuged at 4 °C immediately after collection, and the plasma was separated and stored at −80 °C until analysis could be performed.4)2(SO4)2 or FeCl3 was added into each well of a 96-well plate and warmed to 37 °C. Subsequently, 10 µL of 100 mM N,N′-diethyl-p-phenylenediamine and an aliquot (4 µL) of plasma were added into each well of the 96-well plate and reacted at 37 °C. The reaction product was measured by using an xMark microplate spectrophotometer  with the wavelength, reading mode, and kinetic parameter set at 505 nm, kinetic, and rate, respectively. The results of the Fe-ROMs test were expressed as the rate of increase in OD (mOD/min) during a 3 min period from 2 to 5 min after the addition of the plasma, where 1 mOD/min corresponded to 23.7 µM tert-butyl hydroperoxide. Measurements were run in duplicate and a mean value was used in the analyses (intra-assay coefficient of variability = 3.84%).The amount of hydroperoxides present in a 20 µL volume of plasma was estimated by performing the d-ROMs test using the FRAS4 system according to the manufacturer’s instructions . The results obtained from the d-ROMs test were expressed in arbitrary units called “Carratelli units” (U.CARR). In the Fe-ROMs test, 186 µL of 0.1 M acetate buffer (pH 4.8) containing 100 µM Fe(NH2. The mixture was then stood at room temperature for 10 min and centrifuged (1500× g) for 30 min at 4 °C. The supernatant was ultra-filtered by passing through a 0.45 µm-Millex-HV syringe filter unit, and the filtrate was then stored as the HDL fraction at −80 °C. The stored plasma and HDL fractions were used for the lipoprotein analysis by using a dual detection HPLC system at Skylight Biotech  according to the procedure described by Usui et al. [The plasma was ultra-filtered by using a Millex-HV syringe filter unit . Part of the filtrate was stored at −80 °C as the plasma fraction, and the rest of it was processed to isolate the HDL fraction as reported previously : 0.9 mL i et al. .Any turbidity in the plasma samples was cleared by ultrafiltration through a 0.45-µm Millex-HV syringe filter unit. The cleared plasma sample (0.5 mL) was then separated on an OH pak SB-804HQ column (300 × 8.0 mm) with 0.15 mol/L NaCl at a flow rate of 0.5 mL/min. The column temperature was set at 20 °C, and elution was monitored by absorbance at 260 nm. The column eluate was collected in 0.5 mL fractions and used for the Fe-ROMs test, HDL ELISA assay, and SDS-PAGE analysis.The HDL ELISA assay was done by using an HDL Human ELISA Kit. Briefly, the column eluate (fractions 14–18) was diluted 1:1 with 1× Diluent M; and an aliquot (25 µL) of the diluted sample was added to the wells of a 96-well plate that had been pre-coated with an antibody specific for HDL. After this addition, the assay was done as indicated in the manufacturer’s instructions; and HDL concentrations were calculated from the standard curve.An aliquot (5 µL) of the column eluate was heated at 70 °C for 10 min in a total 40 µL of a Bolt LDS sample Buffer containing Bolt Reducing Agent. It was then applied on a Bolt 4%–12% Bis-Tris Plus gel, and electrophoresed in Bolt MOPS SDS Running Buffer at 200 volts for 35 min. In parallel, 2 µL of the Bio-Rad Precision Plus Protein Dual Color Standards and 5 µL of 1 mg/mL human serum albumin were run. The gels were then washed with ultrapure water, stained for 1 h with Imperial Protein Stain , and destained with ultrapure water. The stained protein bands were detected by using an Image Quant LAS 4000 mini .The plasma was cleared by ultra-filtration using a Millex-GV syringe filter unit . The cleared plasma (0.3 mL) was loaded onto a Protein G Sepharose 4 Fast Flow column (gel volume of 2 mL) and eluted with 10 mmol/L phosphate-buffered saline (pH 7.4) at 0.2 mL/min. The eluate was collected in a total of 18 fractions, each containing 0.2 mL. Seven fractions with high absorbance at 280 nm were collected and concentrated by using an Amicon Ultra-0.5 mL 100K  according to the manufacturer’s instruction, and analyzed by electrophoresis on a Bolt 4%–12% Bis-Tris Plus gel.2+ precipitation method. It was then diluted 1:50 with Assay Diluent, and an aliquot (100 µL) of the diluted HDL was used for the determination of MDA-HDL.Quantitation of MDA-HDL: MDA-HDL was quantitated by using an OxiSelect Human Oxidized HDL ELISA Kit . For this assay, the HDL fraction was first isolated from the plasma by the dextran sulfate-MgPurification of LDL/VLDL: One ml of plasma from a healthy male subject was used for the LDL/VLDL purification. The LDL/VLDL was purified by using an LDL/VLDL Purification Kit , and its activity was determined by performing the Fe-ROMs test after dialysis against a phosphate-buffered saline (pH 7.4).Data were presented as mean ± standard deviation (SD) and subjected to a one-way analysis of variance ANOVA with Tukey HSD post-hoc comparisons. The Pearson’s correlation coefficient was used to identify the association between the levels obtained by the d-ROMs and Fe-ROMs tests. It was also conducted for the association between the levels quantified by the Fe-ROMs test and the MDA-HDL ELISA kit after the HDL fractionation. The analysis was performed using SPSS software version 24 , and the statistical level of significance was set at 0.05.The Fe-ROMs test was demonstrated to be able to assess oxidative stress with ease by using just a small amount of human plasma. Further, oxidized HDL can be estimated from Fe-ROMs levels in plasma. Emerging evidence shows that the anti-atherogenic role of HDL is not simply defined by the plasma HDL-C level but rather by a functional property of HDL. Because the functionality of HDL is affected by the oxidization of HDL components, the measurement of oxHDL must be useful for the assessment of the risk of cardiovascular disease.


Escherichia coli, labeled with a fluorophore, (DCC-EcSSB) was developed and used for this purpose. However, the multiple binding modes of this protein meant that interpretation of DCC-EcSSB fluorescence was potentially complex in terms of determining the amount of ssDNA. Here, we present an improved biosensor, developed using the tetrameric SSB from Plasmodium falciparum as a new scaffold for fluorophore attachment. Each subunit of this tetrameric SSB was labeled with a diethylaminocoumarin fluorophore at a single site on its surface, such that there is a very large, 20-fold, fluorescence increase when it binds to ssDNA. This adduct can be used as a biosensor to report ssDNA formation. Because SSB from this organism has a single mode of binding ssDNA, namely 65–70 bases per tetramer, over a wide range of conditions, the fluorescent SSB allows simple quantitation of ssDNA. The binding is fast, possibly diffusion controlled, and tight (dissociation constant for DCC-PfSSB <5 pM). Its suitability for real-time assays of ssDNA formation was demonstrated by measurement of AddAB helicase activity, unwinding double-stranded DNA.Single-stranded DNA (ssDNA) is a product of many cellular processes that involve double-stranded DNA, for example during DNA replication and repair, and is formed transiently in many others. Measurement of ssDNA formation is fundamental for understanding many such processes. The availability of a fluorescent biosensor for the determination of single-stranded DNA provides an important route to achieve this. Single-stranded DNA binding proteins (SSBs) protect ssDNA from degradation, but can be displaced to allow processing of the ssDNA. Their tight binding of ssDNA means that they are very good candidates for the development of a biosensor. Previously, the single stranded DNA binding protein from  Many cellular processes involving DNA, such as replication, transcription and repair rely on separating double-stranded DNA (dsDNA) to form single-stranded DNA (ssDNA). Measurement of ssDNA formation is fundamental for understanding many such processes, in particular the activity of the large number of helicases that catalyze the strand separation using the energy of ATP hydrolysis. DNA helicase activity can be assayed directly by measuring either the formation ssDNA or loss of dsDNA. Several assays have been developed, mostly based on fluorescence of intercalating dyes, whereby the dye binds to dsDNA preferentially over ssDNA , 2 and bin situ -7-diethylaminocoumarin-3-carboxamide), 6-IATR (6-iodoacetamidotetramethylrhodamine) and 5-IATR (5-iodoacetamidotetramethylrhodamine) were gifts from J. Corrie  –21. dT35l, U.K.) .5’bio-TEG-GGG TAT TGT TTG TTC CCT GAG CGC G 3’) and various reverse primers . Bio-DNAs were gel purified using Qiagen Gel Extraction kit.Biotin-TEG-DNA, linear dsDNA of different lengths but without Chi-sequences, for the AddAB assay, was prepared by PCR from the pCERoriD 4907 bp plasmid  and amplP. falciparum SSB is transcribed and translated from nuclear DNA with a 76-amino-acid, apicoplast-localization sequence (ALS) that is cleaved off once delivered to the apicoplast , L = [DCC-PfSSB], P = [total ssDNA], K = ratio of Kd values for the two SSB species (labeled/unlabeled).Titrations with diethylaminocoumarin-labeled SSBs were done by excitation at 433 nm and emission at 473 nm (both slits at 2.5 nm). For titrations to determine the relative affinities of labeled and unlabeled SSB, dTFmin = fluorescence in the absence of DNA, Fmax = fluorescence of DCC-PfSSB·ssDNA, L = [DCC-PfSSB], P = [total ssDNA] and Kd is the dissociation constant.Titrations at very low [DCC-PfSSB] were fitted to a quadratic binding equation:The fluorescence quantum yields were measured as previously described  using CoThe stopped-flow apparatus was a HiTech scientific SF61MX stopped-flow  with a mercury-xenon lamp. All measurements with DCC-PfSSB and DCC-EcSSB had the excitation at 436 nm and fluorescence emission was measured with a 455 nm cut-off filter (Schott glass). The concentrations given are final concentrations for the components in the mixing chamber. Measurements were repeated as separate experiments three times unless otherwise stated. Kinetic Studio software  was used to record and fit the data. Time courses shown in figures are of representative single measurements. Further data fitting was done using Grafit 7  . Plots oAll fluorescence and kinetic measurements (without helicase) were at 20°C in high salt buffer  except when stated, in which case the low salt buffer had 20 mM NaCl instead of 200 mM.HB (trap) and 2 mM ATP.The AddAB helicase assay for unwinding linear dsDNA was performed on the stopped-flow apparatus at 37°C in buffer containing 25 mM Tris·acetate (pH 7.5), 2 mM magnesium acetate, 1 mM DTT and 10 μM BSA. 1 nM of bio-DNA was incubated with 10 nM streptavidin for 15 min at room temperature. To this 8 nM of AddAB wild type helicase was added and incubated 1 min at room temperature. The mixture was moved to a stopped-flow syringe and incubated for further 1 min at 37°C before rapidly mixing with 200 nM DCC-PfSSB, 100 nM AddA70, a simple model for complete ssDNA binding. As the binding site of PfSSB has been measured as ~65–70 bases k+3(k-2+k+3). At very low concentrations of wtSSB, this pathway is less favoured and the simple dissociative pathway may occur, in which complete dissociation of DCC-PfSSB.ssDNA (step 4) occurs before wtSSB binds to the ssDNA (step 5). Because the concentration of wtSSB is much greater than the labeled SSB, it may be assumed that step 4 is rate limiting and irreversible, so the observed rate constant is k+4. Overall, the observed rate constant for dissociation is, therefore, k+4 + k+2[wtSSB]k+3(k-2+k+3) and the y-intercept in k+4. Because of the tight binding and large signal change with DCC-PfSSB, this measurement could be done at very low concentrations to give a more precise extrapolation to this intercept (-1). From this, the 95% confidence limit gives the range for k+4 from -0.0017 to 0.0015, so the dissociation rate constant as likely to be <0.0015 s-1.A mechanism for dissociation was considered with two distinct pathways  in orderntercept  inset. E70 and DCC-PfSSB is <4.6 pM. Labeling PfSSB reduces its affinity up to ~25-fold as shown by the titrations with a mixture of unlabeled and labeled PfSSB , the first dT35 will preferentially bind away from the fluorophore. However, the second dT35 must fill the remainder of the binding groove. Overall, the three lengths of ssDNA give similar fluorescence enhancement, ~20-fold, on saturation.The titrations support the binding capacity of each tetramer being 65–70 bases. This is shown by the poly(dT) titrations, where the fluorescence increases then plateaus at this ratio of poly(dT) bases to DCC-PfSSB . When dTncements . This isHaving summarized basic binding of ssDNA, properties of DCC-PfSSB will be discussed that differ from DCC-EcSSB in terms of fluorescence and DNA binding. This includes evidence that 65-70-base binding mode is more favoured over 35-base binding with DCC-PfSSB, rather than DCC-EcSSB. In other words even at low salt conditions are possible where there is not a change in binding mode as described below.DCC-PfSSB has a very high fluorescence enhancement on binding ssDNA (20-fold), ~3-fold greater change than DCC-EcSSB (6-7-fold). Diethylaminocoumarin fluorescence depends on its physical environment, in particular on the extent to which the diethylamino moiety is held coplanar to the rings –30. HereAn important point with PfSSB is the extent to which its binding is mainly in the 65-base mode, so simplifying interaction with ssDNA under a range of conditions . As desc70 and dT35 titrations. With dT70 at both high and low salt, the titrations are monotonic, linear responses up to 1:1 ratio, not showing any curvature at low salt that DCC-EcSSB exhibited due to 35-base binding at low ratio. With dT70, the difference in binding of the first and the second DNA molecule is not much changed by the reduction of the ionic strength. In addition, the difference in fluorescent response between binding the first and the second dT35 is only small and both portions of the titration are linear, suggesting that both first and second DNA molecules bind tightly in both conditions. With DCC-EcSSB there were distinct phases for binding of the first and the second dT35 at both high and low salt, suggesting weak binding of the second particularly at low salt . The linear fit had a gradient of 0.107 ± 0.008 μM-1s-1 and intercept of 0.014 ± 0.004 s-1.DCC-PfSSB was premixed with a slight excess of dT(PDF)Click here for additional data file.S1 TableThese were used to change the sequence from the original synthesized gene for (G103C)PfSSB.(PDF)Click here for additional data file.